Two-stage miRNA delivering scaffolds for patient-originated cells to regenerate blood vessels There are about 1.4 million arterial bypass operations annually in the US, but many patients who require arterial bypass procedures do not have suitable vessels for use. Although synthetic materials are frequently used to treat vascular disease, their failure rate, especially in replacing small-diameter vessels, remains high. Induced pluripotent stem cells (iPSCs) have enormous potential for the repair of diseased or traumatized blood vessels. Indeed, we, among the first, have successfully induced iPSC differentiation to smooth muscle cells (SMCs) in culture and on biomimetic 3D scaffolds. However, it takes more than 2 months to generate patient- specific iPSCs and iPSC-derived SMCs and there is potential for oncogenesis. Provocatively, we have established a direct differentiation protocol for SMCs from patient fibroblasts using three determined factors. Also, we found that microRNA-10a (miR-10a) plays an important role in SMC differentiation and helps SMCs maintain the differentiated contractile phenotype. For SMC direct differentiation and future clinical application, an efficient non-viral vector is highly desired. Fortunately, we recently developed a novel hyperbranched polymer vector and a two-stage delivery system to highly efficiently deliver microRNAs and plasmids into cells in a temporally controlled manner. Our long-term goal is to regenerate functional human blood vessels using patient-originated fibroblasts. The key to functional blood vessel regeneration using fibroblasts is the in situ direct differentiation and maintenance of the contractile phenotype of the vascular SMCs. In this proposal, we hypothesize that sustained and highly efficient miRNA/plasmid delivery into fibroblasts and SMCs on a 3D nanofibrous scaffold will regenerate a contractile SMC-laden functional vascular graft. The specific aims of this project are: 1) Determine the mechanistic roles of miR-10a, HDAC4 and associated signaling pathways in fibroblast/SMC transdifferentiation; 2) Develop anatomically-designed nanofibrous scaffolds using a reverse 3D printing technology and immobilize sustained two-stage miRNA/plasmid delivery system on the scaffolds; 3) Evaluate engineered vascular scaffold in vitro and in vivo. By accomplishing these specific aims, we will improve mechanistic understandings of fibroblast/SMC transdifferentiation how to maintain the SMC contractile phenotype in the vascular scaffold and develop key miRNA/DNA delivery and 3D tissue engineering technologies to advance the therapeutic utility of patient-originated cells for human vascular regeneration.